Artificial genetic systems have been the targets of organic chemists for more than a decade.1,2,3,4 They have recently attracted attention in the scientific literature5,6,7 and the science press.8,9 One class of these retains the two rules of complementarity that characterize standard Watson-Crick base pairing: (i) size complementarity, whereby a large purine pairs with a small pyrimidine, and (ii) hydrogen bonding complementarity, whereby hydrogen bond donors complement hydrogen bond acceptors.10 
With three hydrogen bonds joining the paired nucleobases, twelve nucleobases and six mutually exclusive hydrogen bonding patterns are possible (FIG. 1). These were preared some time ago by Benner as part of an Artificially Expanded Genetic Information System (AEGIS). Diagnostics products based on AEGIS have been approved from the Food and Drug Administration for clinical use to monitor viral load in patients living with HIV and hepatitis C.11 
As is widely recognized by those skilled in the art, it would be useful to be able to amplify the oligonucleotides containing non-standard components in a process analogous to the well known polymerase chain reaction. This might even allow an artificial genetic system to be incorporated into a living cell.12 This would in particular be useful for doing in vitro selection, Selex, and in vitro evolution of an expanded genetic information system for the purpose of creating new catalysts, ligands, and receptors.
Some time ago, Benner patented a process for doing this [U.S. Pat. No. 5,965,364]. This process has never, however, been executed. This is because the specification of U.S. Pat. No. 5,965,364 did not disclose a polymerase that incorporates a non-standard base pair into a duplex with sufficient efficiency and fidelity to support such a process. This was illustrated by Johnson et al.13, who attempted to incorporate isocytosine and isoguanine into a PCR-type process. As their publication shows, the non-standard component is not retained, to an extent of at least 90% over 5 cycles, in the product. This can be taken as a crude metric for the utility of a PCR process that incorporates a non-standard nucleobase.
The difficulty in incorporating AEGIS components into a PCR process may be due to difficulties in the efficiency by which polymerases interact with unnatural nucleoside triphosphates. Many enzymes work well with AEGIS components, including kinases, ligases, and even ribosomes.14 Polymerases, in contrast, accept many non-standard components of DNA only inefficiently, judging by rate, processivity, fidelity, or some combination of these.15 These inefficiencies need not prevent the utility of polymerase-based incorporation of AEGIS components in single pass experiments, and may not be apparent with standing start experiments, where the non-standard triphosphate is the first nucleotide to be added to a primer, or a running start experiment, where the polymerase adds standard nucleotides before it is challenged to incorporate a non-standard nucleotide.
Without expressing an opinion about why Johnson et al. failed to generate a useful PCR process, it would nevertheless be useful to be able to amplify via the polymerase chain reaction such oligonucleotide analogs (herein referred to as “oligonucleotides”, despite their not being standard, natural oligonucleotides), so that a small number of starting oligonucleotides can generate multiple product oligonucleotides, through the copying of oligonucleotides containing AEGIS components, and then copying the copies.
While it is recognized that such processes invariably introduce some mutations, and that some of the daughter oligonucleotides will not have the exact identical sequence as the original oligonucleotide (and indeed, this evolution is useful in some contexts), PCR amplification of these oligonucleotides would be most useful if the level of mutation is lower rather than higher, preferably less than a 5% loss of the non-standard nucleobase per cycle, and more preferably less than a 2% loss of the non-standard nucleobase per cycle, and most preferably retaining 90% of the AEGIS component after 5 cycles.
The object of the instant invention is to provide a process that does PCR amplification of AEGIS-containing oligonucleotides with two specific AEGIS components, one presenting the donor-acceptor-donor hydrogen bonding pattern on a pyrimidine skeleton, hence described as the pyDAD system, herein implemented on the diaminopyrimidine heterocycle, the other presenting the hydrogen bond acceptor-donor-acceptor hydrogen bonding pattern on a purine skeleton, hence described as the puADA system, herein implemented on the xanthosine heterocycle.
Also obvious to one of ordinary skill in the art, to be useful, the PCR process exploiting AEGIS components must be associated with a sequencing tool that allows the practitioner of the art to assess the extent to which the AEGIS component has been retained in the PCR products. It is the object of the instant invention to provide a provide a process for doing so.